INTRODUCTION

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BRIEF EPISODES of ischemia-reperfusion protect the myocardium from the damage induced by subsequent more prolonged ischemia, and this protection is called "ischemic preconditioning" (IP). Whereas the first window or early phase of protection is short lived (classic IP), a second window or late phase of protection appears about 24 h after the preconditioning ischemia-reperfusion. The signal transduction pathway leading to protection in the late phase shares many of the steps identified for the early phase protection (18).

Circulating and cardiac tumor necrosis factor- (TNF-) concentrations increase in response to myocardial ischemia-reperfusion within minutes, most likely by release from macrophages, monocytes, and mast cells (2, 6). Classic IP decreases cardiac and circulating TNF- concentrations during the sustained ischemia and reduces myocardial infarct size in rabbits (3, 14). Although a causal role between reduced TNF- and reduced infarct size was not established, these studies suggest a deleterious role of TNF- during the sustained ischemia. This assumption is further supported by the fact that pretreatment with TNF- antibodies before ischemia-reperfusion reduced infarct size in anesthetized rabbits (11).

On the other hand, inflammatory cytokines such as TNF- are also involved in triggering IP, because pretreatment with TNF- antibodies abolished infarct size reduction achieved by late IP in a rat model of myocardial infarction (22), and genetic ablation of TNF- abolishes infarct size reduction by classic IP in mice (19).

The exact role of TNF- in the signal cascade of classic IP has not yet been established. The aim of the present study was therefore to investigate whether or not pretreatment with TNF- antibodies interferes with the infarct size reduction by classic IP.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Surgical preparation. Chinchilla bastard rabbits (3.0-3.6 kg; mean 3.2 kg) were anesthetized with an intramuscular injection of ketamine hydrochloride (50 mg/kg) followed by an intravenous infusion of propofol (6-10 ml/h). Rabbits were then intubated with an endotracheal tube (5 mm internal diameter), placed in the supine position on a table, and ventilated using positive pressure. Ventilation was maintained by using a Dräger UV-2 ventilator with 30% oxygen-70% room air. Arterial blood gases were monitored frequently in the initial stages of the preparation until stable and then periodically monitored throughout the study (Radiometer ABL-510; Copenhagen, Denmark). Tidal volume was adjusted to maintain arterial PCO₂ between 30 and 40 mmHg and PO₂ between 120 and 150 mmHg. Rectal temperature was continuously monitored, and hypothermia was prevented by using a heating pad. The left common carotid artery was cannulated with a polyethylene catheter, and its tip was advanced into the aortic arch for blood pressure measurement and arterial blood withdrawal. Both common jugular veins were cannulated with polyethylene catheters for the administration of saline and drugs. The heart was exposed in a pericardial cradle through a left thoracotomy, and a 4-0 prolene suture was placed around the anterolateral branch of the left circumflex coronary artery, midway between the atrioventricular groove and the apex. The suture was passed through a soft plastic tube to form a snare for coronary artery occlusion. Cyanosis and ECG changes were considered to indicate effective coronary artery occlusion. The animals were heparinized with 2,000 IU heparin sodium. At the end of each experiment, the ligature around the anterolateral branch of the left circumflex coronary artery was retightened, and 15 ml of methylene blue solution were injected as a bolus into the jugular vein until the heart not supplied by the occluded coronary artery turned blue. The rabbits were killed immediately, and the heart was removed.

Experimental protocol. Rabbits in group 1 (n = 7) were subjected to 30 min of coronary artery occlusion and 180 min of reperfusion. Rabbits in group 2 (n = 6) were subjected to 5 min of coronary artery occlusion and 10 min of reperfusion before the 30-min coronary artery occlusion and 180-min reperfusion. Rabbits in group 3 (n = 6) received anti-murine TNF- sheep antibodies 1 h before the 30-min coronary artery occlusion and 180-min reperfusion. The protocol in group 4 (n = 6) was identical to that in group 2, except that rabbits received anti-murine TNF- sheep antibodies 1 h before the first preconditioning coronary artery occlusion.

Hemodynamics. Maximal and mean aortic pressures and heart rate were recorded on an eight-channel recorder (Gould MK 200A) and stored directly on the hard disk of an AT-type computer. Data were taken at baseline, at 20 min coronary artery occlusion, and at 60 reperfusion.

Circulating TNF- concentration. Blood was collected from a common jugular vein, and serum was prepared by centrifugation and stored at 70°C. Blood samples were obtained at baseline at 20-min coronary artery occlusion and 60-min reperfusion in groups 1 and 3 and at baseline at 5 min of the preconditioning ischemic period, at 20 min of the subsequent coronary artery occlusion, and at 60 min of reperfusion in groups 2 and 4. Circulating TNF- concentration was determined by using a cytolytic cell assay [mouse fibrosarcoma cell line WEHI 164, clone 13, kindly donated by Dr. T. Espevik, Oslo, Norway (5)]. The WEHI cells (2 × 10⁴ cells/well) were incubated with serial dilutions of supernatant in microtiter plates (Nunc, 1:8 to 1:256) at 37°C for 18 h. During this time, recombinant mouse TNF- added in parallel caused 50% cell lysis at a concentration of 10 pg/ml. 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (500 µg /well) was then added. The reaction was stopped after 4 h by addition of 5% formic acid in isopropanol, which solubilizes the remaining cells and MTT. The content of reduced MTT, which has been previously shown to be a quantitative measure of TNF- concentration (5), was read in a micro-ELISA autoreader (MR 580, Dynatech Laboratories; Alexandria, VA). The concentration of TNF- was expressed in units per milliliter; 1 unit is the reciprocal of the dilution necessary to cause 50% cell destruction (5).

TNF- antibodies. Serum of a sheep immunized against recombinant murine TNF- was used to pretreat rabbits in groups 3 and 4. The antiserum was found to be cross reactive with rabbit, rat, and human TNF-, but no other cytokine was tested. In in vitro experiments using rabbit recombinant TNF- (BD Pharmingen), the antiserum neutralized the rabbit recombinant TNF- concentrations of 40, 80, and 100 pg/ml to 89%, 98% and 92%, respectively. Circulating TNF-, determined by using WEHI-163 clone cytotoxic activity assay (5), was neutralized to a maximum of 85% at antiserum dilutions of 1:100 (2 mg/ml) after 1 h of incubation. The concentration of antiserum for in vivo administration was calculated to be 25 mg/kg antiserum, which was infused intravenously 1 h before ischemia. This concentration of antiserum abolished the ischemia-induced increase in serum TNF- concentration in anesthetized dogs (4).

Area at risk and infarct size. At the end of each experiment, the heart was removed and cut from base to apex into five slices of 3- to 4-mm thickness each. All slices were photographed, and images were stored directly on an optical disk. The tissue slices were then stained in 1.0% triphenyltetrazolium chloride (TTC, Sigma; Deisenhofen, Germany) and 8% dextran (77,800 mol wt) for 20 min at 37°C. After TTC staining, the slices were again photographed, and images were stored on an optical disk. Area at risk was determined by negative staining with methylene blue. Red-stained viable tissue was distinguished from the infarcted pale, nonstained necrotic tissue. Area at risk and infarct size were measured by computer-assisted planimetry of the calibrated pictures and analyzed with the Adobe Photoshop 3.0 software. Area at risk was expressed as the percentage of the left ventricle, and infarct size was expressed as the percentage of the area at risk.

Statistics. Statistical analysis was performed with Sigma Stat software (Jandel Scientific; San Rafael, CA). All data are reported as means ± SD. Systemic hemodynamics and serum TNF- concentrations were compared using two-way analysis of variance. When a significant overall effect was detected, single mean values were compared using Bonferroni's method. Comparisons of area at risk and infarct size were made by one-way analysis of variance. Data were considered statistically significant at a P value <0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mortality. Two rabbits of group 1, one rabbit of group 2, and one rabbit of group 4 died from ventricular fibrillation during coronary artery occlusion.

Hemodynamics and infarct size. Heart rate and maximal and mean aortic blood pressures did not change throughout the protocol and were not different among groups (Table 1).

View this table: [in this window] [in a new window]   Table 1.   Systemic hemodynamics and serum TNF- concentration

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Infarct size. AAR, area at risk. Data are means ± SD; n = 7 rabbits in group 1 and n = 6 rabbits in groups 2-4. Group 1, control; group 2, preconditioned; group 3, pretreated with tumor necrosis factor- (TNF-) antibody; group 4, pretreated with TNF- antibody and preconditioned. *P < 0.05 vs. group 1.

Circulating TNF- concentration. Circulating TNF- concentration was increased during coronary artery occlusion (Table 1), and this increase was maintained throughout reperfusion (not significant) in group 1. In group 2, circulating TNF- concentration was increased at 5 min of the preconditioning ischemia from 574 to 1,140 U/ml. During the subsequent coronary artery occlusion and reperfusion, circulating TNF- concentration was, however, not different from the concentration at baseline. In groups 3 and 4, circulating TNF- concentrations did not increase during ischemia-reperfusion.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Circulating TNF- concentrations increased in group 1 during the sustained ischemia and in group 2 during the initial cycle of 5-min preconditioning ischemia and 10-min reperfusion, confirming the results of our previous study (3). Myocardial ischemia-reperfusion increased the circulating and cardiac TNF- concentrations, and IP abolished these increases in circulating as well as in cardiac TNF- concentrations (3, 14). Given the very short ischemic time interval (minutes), it is most likely that the alterations in circulating and cardiac TNF- were secondary to the release of preformed TNF- from blood-borne and/or resident macrophages and mast cells rather than to de novo synthesis by cardiomyocytes.

Pretreatment with TNF- antibodies abolished the increase in the circulating TNF- concentration throughout the experimental protocol in both nonpreconditioned and preconditioned rabbits, and the circulating TNF- concentration during the sustained ischemia and the subsequent reperfusion was similar in preconditioned and TNF- antibody-treated rabbits. The close correlation between circulating TNF- concentration during the sustained ischemia and infarct size suggests a deleterious role of TNF- in acute myocardial infarction, and this hypothesis is supported by the fact that neutralization of TNF- by TNF- antibodies (11) or its absence in TNF- knockout mice (12) is cardioprotective. The mechanisms by which TNF- causes myocardial injury in acute myocardial infarction include direct cytotoxity, increased oxidative stress, and activation of matrix metalloproteinases, which are capable of degrading the components of the extracellular matrix (9, 13).

In contrast to such a deterious role of TNF- during sustained acute ischemia, previous studies suggested that TNF- is involved in triggering during the late phase (22) and the early phase (19) of IP, thereby reducing infarct size during a subsequent more prolonged ischemia. However, for the late phase of protection, both TNF- and interleukin (IL)-1 are required, because only combined blockade of both TNF- and IL-1 abolishes the late phase of the protection of IP (22). Thus TNF- and IL-1 act in parallel to initiate delayed IP. A similar redundant pathway was demonstrated in pigs (21) and rats (7, 20) for protein kinase C and protein tyrosin kinases; in both species only combined blockade of both protein kinases abolished infarct size reduction by IP, whereas blockade of either kinase alone did not interfere with the protection of IP.

Additional evidence that TNF- might act as a trigger of classic IP is supplied by Lecour et al. (10), who demonstrated that TNF-, given before the sustained ischemia, mimicked IP. However, exogenous application of a substance does not necessarily provide informations about its endogenous role. For example, nitric oxide when applied exogenously mimicks the infarct size reduction of IP (15); however, blockade of endogenous nitric oxide does not abolish the protection of IP (15, 16). The mechanism of cardioprotection by TNF- is not fully understood but might be related to mitochondrial ATP-sensitive potassium channel activation (10).

With the use of TNF- antibodies in the present study, rather than genetic ablation of TNF- (19) or administration of TNF- before the index ischemia (10), we were not able to support or dismiss the above hypothesis because the dominant effect of TNF- antibodies was the infarct size reduction per se achieving the same magnitude of protection seen with one cycle of 5 min of preconditioning ischemia and 10 min of reperfusion.

A previous study in rabbits had suggested that IP is a dose-dependent phenomenon (17). We have therefore tried to increase the protection of IP by increasing the number of preconditioning ischemia-reperfusion cycles from one to four with the aim to possibly attenuate this greater protection with TNF- antibodies. However, in agreement with other prior studies (1, 8), a similar infarct size was obtained in three additional rabbits, which underwent four cycles of 5-min preconditioning ischemia with subsequent 5-min reperfusion before 30-min coronary artery occlusion and 180-min reperfusion (14 ± 5%) as in rabbits with only a single cycle of preconditioning ischemia-reperfusion (13 ± 7%).

Thus the present study can neither prove nor disprove the causal involvement of TNF- in the signal cascade of classic ischemic preconditioning.